A nitrogen isotopic shift in fish otolith–bound organic matter during the Late Cretaceous

Significance The geochemistry of fossils is used to reconstruct the ocean environment in Earth history. Our study shows the potential of organic matter within the biomineral matrix of fossil fish ear stones (otoliths) for this purpose. We report a change in nitrogen isotope ratios of fossil otoliths during the Late Cretaceous along the eastern North American continental shelf. We favor interpretation of the change as a global ocean signal, suggesting that the ocean’s oxygen-deficient zones expanded during the climate cooling of the Late Cretaceous. The large and coherent signal observed in this study bodes well for fossil otolith-bound δ15N as a window into past oceanic change, available even when open ocean sedimentary deposits are lacking.

The nitrogen isotopes of the organic matter preserved in fossil fish otoliths (ear stones) are a promising tool for reconstructing past environmental changes.We analyzed the 15 N/ 14 N ratio (δ 15 N) of fossil otolith-bound organic matter in Late Cretaceous fish otoliths (of Eutawichthys maastrichtiensis, Eutawichthys zideki and Pterothrissus sp.) from three deposits along the US east coast, with two of Campanian (83.6 to 77.9 Ma) and one Maastrichtian (72.1 to 66 Ma) age.δ 15 N and N content were insensitive to cleaning protocol and the preservation state of otolith morphological features, and N content differences among taxa were consistent across deposits, pointing to a fossil-native origin for the organic matter.All three species showed an increase in otolith-bound organic matter δ 15 N of ~4‰ from Campanian to Maastrichtian.As to its cause, the similar change in distinct genera argues against changing trophic level, and modern field data argue against the different locations of the sedimentary deposits.Rather, the lower δ 15 N in the Campanian is best interpreted as an environmental signal at the regional scale or greater, and it may be a consequence of the warmer global climate.A similar decrease has been observed in foraminifera-bound δ 15 N during warm periods of the Cenozoic, reflecting decreased water column denitrification and thus contraction of the ocean's oxygen deficient zones (ODZs) under warm conditions.The same δ 15 N-climate correlation in Cretaceous otoliths raises the prospect of an ODZ-to-climate relationship that has been consistent over the last ~80 My, applying before and after the end-Cretaceous mass extinction and spanning changes in continental configuration.

fossil fish otoliths | nitrogen isotopes | Late Cretaceous | marine biogeochemistry
In the Late Cretaceous, global climate underwent a transition from the warm "hothouse world" that characterized much of the Cretaceous to a cooler climate that persisted into the Early Cenozoic.As reconstructed from benthic and planktonic foraminiferal oxygen isotopes (1,2), the Late Cretaceous cooling began in the late Campanian (~75 Ma) and extended into the early Maastrichtian (72 to 69 Ma), apparently associated with declining atmospheric CO 2 (3,4).The cooling was observed in deep waters (1,2,5), surface waters (6)(7)(8), and the inland Western Interior Seaway (9).Sea level reconstructions from the Atlantic Coastal Plain exhibit rapid sea level fluctuations of up to 50 m in the Late Cretaceous, suggestive of ice volume fluctuations (10,11).
We know relatively little about the biogeochemical and ecosystem changes that occurred in response to the substantial global cooling of the Late Cretaceous, in the ocean or on land.Nitrogen (N) isotopes (the 15 N/ 14 N ratio, or δ 15 N) have the potential to provide such information, especially in marine settings (δ 15 N = (( 15 N/ 14 N) sample /( 15 N/ 14 N) AIR, N2 -1)*1000).N isotope reconstructions can provide insights into the δ 15 N "baseline", which is controlled by global and regional ocean biogeochemical processes (12).To date, N isotope studies of the Late Cretaceous have focused on the δ 15 N of bulk sedimentary N (13,14).The δ 15 N of bulk sediments can provide important information on the N cycle (15), but, in most sedimentary environments, the preservation of bulk sedimentary organic matter δ 15 N is questionable for recent sediments (16), let alone for millions-of-years-old Mesozoic deposits.

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In this study, we analyzed δ 15 N oto from Late Cretaceous fossil otoliths along the Atlantic Coastal Plain of the United States east coast (Fig. 1).Fossil otoliths were recovered from sediments within the Woodbury Formation (New Jersey, USA, Campanian age), Tar Heel Formation (North Carolina, USA, Campanian age), and Severn Formation (Maryland, USA, Maastrichtian age).We selected the otoliths of Eutawichthys maastrichtiensis, Eutawichthys zideki, and Pterothrissus sp.These species were the three most abundant in the sedimentary deposits across the time periods of interest.The Eutawichthys species occur over the continental shelf at ≤200 m depth, while Pterothrissus sp.usually occurs at 100 to 400 m but can be found at shallower depths (39).Thus, these species should record open shelf conditions as opposed to small-scale coastal signals.We find that δ 15 N oto can be measured robustly in these >66-My-old deposits.Moreover, coherent δ 15 N oto changes are observed that bear on the biogeochemical cycles of the ocean over the changing climate of the Late Cretaceous.

Results
The uncertainty in the δ 15 N oto analysis is far lower than the variation among otoliths, taxa, and deposits.Replication of individual fossil otoliths yielded a δ 15 N oto SD (1σ) of 0.22 ± 0.21‰, which is comparable to the long-term δ 15 N variability of an in-house coral standard (0.29‰).The blank N size for δ 15 N oto analyses is between 0.24 and 0.62 nmol N. The blank contribution is generally less than 5% of total sample N (0.3 to 5.2%).
Mineralogical identification on a subset of sample fossil otoliths, including otoliths of both Eutawichthys spp.and Pterothrissus sp., indicates that all have maintained aragonitic mineralogy (SI Appendix, Figs.S8 and S9).This is despite the microscopic characterization of many of the E. zideki otoliths as being poorly preserved (SI Appendix, Fig. S8).In our cleaning tests, δ 15 N oto is not sensitive to whether the otoliths were ground prior to cleaning (Fig. 2) or to the reagent used for oxidative cleaning (SI Appendix, Table S1).The δ 15 N oto values of qualitatively characterized "well" and "poorly" preserved otoliths also cover a similar range (SI Appendix, Fig. S4 and Table S2).Moreover, across the geologic deposits, δ 15 N oto was similar between well-preserved and poorly preserved Eutawichthys spp.otoliths (10.9‰ vs. 10.6‰,11.4‰ vs. 10.7‰, and 15.4‰ vs. 14.3‰ for Woodbury, Tar Heel, and Severn Formations, respectively; Fig. 3).
Otolith N content is similar between the two investigated species in the genus Eutawichthys (40.5 ± 4.0 μmol N/g in E. maastrichtiensis and 38.2 ± 7.6 μmol/g in E. zideki), while the otolith N content of Pterothrissus sp. is distinct (23.5 ± 2.4 μmol/g) (Fig. 3).For each taxon, the otolith N content is comparable across all deposits, yielding similar taxon-level differences in all three deposits (Fig. 3).
The δ 15 N oto values of all species are similar to one another for the Campanian-age Woodbury Formation (New Jersey) and Tar Heel Formation (North Carolina) (Fig. 3).Eutawichthys spp.δ 15 N oto is 10.77 ± 0.98‰ in the Woodbury Formation and 11.06 ± 1.06‰ in the Tar Heel Formation; the Pterothrissus sp.δ 15 N oto is 10.89 ± 0.83‰ in the Woodbury Formation and 11.28 ± 0.75‰ in the Tar Heel Formation.Within a formation, the mean δ 15 N oto of genera Eutawichthys and Pterothrissus are not significantly different, regardless of apparent preservation state (Student's t test, P = 0.76 in Woodbury Formation, P = 0.58 in Tar Heel Formation, P = 0.84 in Severn Formation).Notably, however, the δ 15 N oto in the Severn Formation (of Maastrichtian age) is clearly higher than that in the two Campanian formations (Fig. 3; Student's t test, P ≪ 0.01 for mean δ 15 N oto of all Maastrichtian otoliths comparing to all Campanian otoliths).This δ 15 N oto increase is observed in both Eutawichthys spp.(10.89 ± 1.02‰ in Campanian, 14.87 ± 1.25‰ in Maastrichtian) and Pterothrissus sp.(11.08 ± 0.79‰ in Campanian, 14.78 ± 0.80‰ in Maastrichtian).

Discussion
Insights into Otolith-Bound N from N Content and Cleaning Tests.
Modern otoliths are characterized by different N contents across taxa (30,35,40), which is also observed in our study of fossil otoliths.The fossil otoliths of one genus in our study, Eutawichthys, have on average higher N content than most modern otoliths from extant species studied so far, whereas the fossil otolith N content of Pterothrissus sp.falls within the typical modern range (30,35).Notably, the taxonspecific N contents of fossil otoliths, including their intertaxonomic differences, are consistent across all three deposits (Fig. 3B).These findings are consistent with the otolith-bound N being native to the otolith and preserved in these geologic settings.
The cleaning tests offer further insight into the fossil otolith-bound N (Fig. 2).Whether the analyzed whole otoliths were 1) cleaned, 2) cleaned and ground, or 3) cleaned, ground, and recleaned, N content was remarkably similar, as was δ 15 N oto (Fig. 2A).If the organic matter (OM) in question was present dominantly as accumulations in the pore spaces within otoliths (orange specs in gray matrix in Fig. 2B), then N content should have declined upon grinding and recleaning.The lack of change in N content (and δ 15 N oto ) with recleaning argues that the OM is intracrystalline or micro-intercrystalline, a distribution more consistent with a fossil-native origin than with diagenetic incorporation.
Additionally, the otolith N content of each taxon is independent of which cleaning reagent was used (SI Appendix, Fig. S3 and Table S1).In the case of using persulfate reagent for cleaning, this is the same reagent used, after dissolving the biomineral in acid, for N oxidation to nitrate in δ 15 N analysis.Therefore, the survival of this OM through the cleaning process can only be explained by physical protection by the biomineral matrix, as opposed to OM survival due to chemical recalcitrance (SI Appendix, Fig. S3A and Table S1).The fossil-bound N must be dominantly occurring within the otolith grains, again pointing to residence of the OM in the least diagenetically accessible components of the biomineral structure and, thus, a fossil-native origin.
Finally, we observe similar N content and δ 15 N oto between well-preserved and poorly preserved Eutawichthys spp.otoliths (Fig. 3 and SI Appendix, Fig. S4), implying that postdepositional alteration to the organic N within these fossil otoliths was unimportant in all three deposits.The slightly lower N content could be due to loss of a small proportion of the otolith-bound organic N when it was exposed to diagenesis.However, there is no evidence of any N isotopic change (SI Appendix, Table S2), implying that any exposure of fossil-bound N leads to its complete loss, consistent with its dominant occurrence as peptide and/or free amino acid N (35), which should be diagenetically labile.These A B Fig. 2. (A) Otolith N content (blue circles) and δ 15 N oto measurements (orange circles) for well-preserved otoliths from the cleaning test.Each group represents a different cleaning treatment: (from Left to Right) uncleaned whole otoliths, cleaned whole otoliths, ground otolith powder from the cleaned whole otoliths, and ground otolith powders with a second cleaning/recleaning.The lower bound of the δ 15 N oto axis range (6‰) is approximately the lowest possible δ 15 N oto for a modern fish in this region, taken as the suspended PN δ 15 N (3‰) in the region plus 3‰ for the trophic level δ 15 N increase.(B) Expected otolith N content changes for different cleaning steps.The colors used for otolith carbonate matrix represent the scenarios without fossil-native organic matter (gray) and with intracrystalline and/or micro-intercrystalline fossil-native organic matter (green).findings conform with cleaning experiments on other fossil types (41).All told, both the methodological tests and the results from the different deposits point to otolith-bound OM as a unique window into ancient organisms and their environments.
Causes of Variation in δ 15 N oto .Within a formation, the intrataxon δ 15 N oto SD are between 0.75 and 1.25‰ (Fig. 3).This variability is not clearly different from the variability observed from the modern butterfish otoliths (0.99‰; Fig. 1), even though the modern otoliths are all from the same ecosystem.Considering that the fossil otoliths were collected from geologic formations that span millions of years and cover large coastal regions, the formation-level variability is remarkably small.Similar δ 15 N oto variability across formations also suggests similar degrees of ecological and temporal averaging for all deposits.Regardless, the within-deposit variability in δ 15 N oto is minor compared to the δ 15 N oto shift of ~4‰ (Fig. 4) from the Campanian formations to the Maastrichtian formation.The δ 15 N oto increase is consistent across all three species (Figs. 3 and 4).It could be attributed to a) an increase in trophic level of both Eutawichthys spp.and Pterothrissus sp.fishes (34) and/or b) an increase in "baseline" δ 15 N, that is, the δ 15 N of the photosynthetic OM available for consumption by higher trophic levels (12,15).We consider both potential explanations below.
Changes in Fish Trophic Level.Fish tissue δ 15 N [which is recorded by δ 15 N oto ; (30)] varies with trophic level, with higher trophic level fishes having a higher δ 15 N. Trophic level can vary based on prey availability and prey preferences of the fish, even within a species (43).Modern fishes of Albulidae and Berycidae, the taxonomic families of Pterothrissus sp. and Eutawichthys spp., respectively, both consume diel-migrating zooplankton, worms, and/or small fishes (44,45).As there is no known major ecosystem turnover during the Campanian-to-Maastrichtian interval, the likelihood of fundamental dietary change in these fishes is low.Another potential driver of trophic level shift is fish size, as larger fishes can occupy higher trophic position within a given species (46,47).However, the ranges of fish size estimated from otolith weight greatly overlap between the Campanian and Maastrichtian (SI Appendix, Fig. S7).Moreover, there is no significant correlation between δ 15 N oto and fossil otolith weight, whether considering all otoliths or only the well-preserved otoliths (SI Appendix, Fig. S7).
Arguably the strongest evidence against trophic change as the cause of the Campanian-to-Maastrichtian δ 15 N oto increase is the similarity of the δ 15 N oto increase observed in all three taxa.If the δ 15 N oto increase were due to trophic level change, both Eutawichthys species and the Pterothrissus species would need to have altered their diets concomitantly.In addition, the 4‰ amplitude of the δ 15 N oto increase would correspond to an increase in one to two trophic levels, depending on the trophic discrimination factor [(48) and references therein].Such a large increase would be extraordinary for species in separate families generating identifiably distinct otoliths, and it would also require significant restructuring of marine food webs between the Campanian and Maastrichtian.There are no obvious drivers for such a dramatic change in fish diet between studied time periods.Thus, the δ 15 N oto change most likely records a Campanian-to-Maastrichtian increase in baseline δ 15 N along the continental shelf on the mid-Atlantic western margin.
On-Shelf Baseline δ 15 N Gradients.Along the modern mid-Atlantic western margin at the depth ranges believed to be preferred by the taxa in our study, there is little evidence for a substantial along-shore δ 15 N gradient.The δ 15 N of multiple N pools does not exhibit a latitudinal change along the continental shelf between 36°N and 44°N (Fig. 1A), implying little latitudinal variation in the δ 15 N of the nitrate supply to the shelf ecosystem.The offshore nitrate δ 15 N decline (Fig. 1B) reflects the well-documented low-δ 15 N nitrate pool of the subtropical gyre thermocline, the low density of which prevents direct exchange with the shelf (49).The sporadic elevation in nitrate δ 15 N in some water samples from the shelf (Fig. 1B) reflects partial assimilation of nitrate, not variation in the δ 15 N of the nitrate supply, as signaled by the lower nitrate concentrations of these samples (SI Appendix, Fig. S2).Near the shelf-slope break, the nitrate δ 15 N increase from south to north is 1‰ or less over the latitudinal range of our data (50).These modern observations aside, the highest Cretaceous otolith δ 15 N measurements are from Severn Formation otoliths in Maryland, located between the New Jersey and North Carolina deposits, while the otoliths from these northernmost and southernmost locations exhibit similar δ 15 N oto (Fig. 3).All of these observations argue against a role for an along-shore gradient in the δ 15 N differences among the three deposits.
A small outer-shelf-to-coast increase in δ 15 N (of 1 to 2‰) is observed in particulate nitrogen (PN), zooplankton, and fish tissues on the modern Northeast coast (51)(52)(53).This may arise in part from fixed N inputs at the coast contrasting with lower δ 15 N of nitrate from the open North Atlantic (Fig. 1B).However, the magnitude of this δ 15 N gradient is known to be attenuated at higher trophic levels for pelagic fishes, likely due to the transport of plankton and the movement of nekton (53).The Cretaceous species investigated here are expected to be found at various shelf depths and certainly not restricted to nearshore (39,42).Therefore, cross-shelf δ 15 N variation, in isolation, is also an unlikely explanation for the ~4‰ increase δ 15 N oto from Campanian to Maastrichtian.
Baseline δ 15 N Change at the Regional or Global Scale.Studies of fossil-bound N isotopes have found evidence of major shifts in baseline δ 15 N through the Cenozoic, on time scales from millions of years (54,55), to orbital cycles (56), to millennial timescales and shorter (57,58).These prior findings are believed to reflect changes in the ocean N cycle on a regional and/or global basis.Moreover, as discussed above, we find little evidence for a strong along-shelf or outer-shelf-to-coast baseline δ 15 N gradient across the latitudes of interest (Fig. 1).Thus, the δ 15 N oto elevation of ~4‰ observed in the Maastrichtian relative to the Campanian likely reflects a baseline δ 15 N change on a scale greater than that of along-and cross-shelf gradients.
Baseline δ 15 N is directly related to the δ 15 N of the shallow subsurface nitrate that is mixed or upwelled into the euphotic zone (22)(23)(24)59).On the western margin of the North Atlantic, the tilted pycnocline exposes nitrate from below the thermocline that underlies the subtropical gyre (49).This subthermocline nitrate is ~4.8‰, similar to the δ 15 N of deep ocean nitrate (60).In the open ocean, the center of the gyre is characterized by thermocline nitrate with a lower δ 15 N (~3‰), due to the remineralization of newly fixed N transported from the south (61).The δ 15 N of the regional nitrate supply to the continental shelf ecosystem could be affected by the strength of N 2 fixation in the North Atlantic and/or changes in the exchange between the gyre thermocline and the shelf, such as through Gulf Stream changes.
N 2 fixation appears to be sensitive to temperature, tending to increase at higher temperature (62).Thus, one might argue that the lower δ 15 N oto of the Campanian was due to the higher temperature of the Campanian causing an increase in N 2 fixation.However, on a global basis and over time scales of thousands of years and longer, N 2 fixation must balance ocean N loss, which dominantly occurs by denitrification (63).Evidence from both the modern ocean and paleoceanographic studies indicate that N 2 fixation responds to N loss rates, through the N-depleted, P-bearing surface waters produced by N loss (61,63).While temperature changes may alter the surface ocean distribution of N 2 fixation, we know of no reason that the Campanian-to-Maastrichtian cooling would have shifted N 2 fixation from the North Atlantic to other regions.
The nitrate supply δ 15 N might change due to changes in regional circulation.For example, a weaker margin-to-gyre surface density gradient may have increased the input of the lower-δ 15 N nitrate of the subtropical gyre thermocline to the margin surface waters (64).However, given modern isotopic gradients in the region, the maximum δ 15 variation due to such a change is ~2.5‰(49).Thus, even a total lack of margin-to-gyre density gradient during the warmer Campanian transitioning to the modern situation during the Maastrichtian would be insufficient to explain the 4‰ increase in δ 15 N oto .
Last, changes in the sedimentary denitrification on the continental shelf can be driven by the sea level change.Eustatic sea level reconstructions in the Late Cretaceous suggest fluctuations of up to 40 m (10,11), with the possibility that reduced continental shelf area decreased sedimentary denitrification during the colder Maastrichtian.Sedimentary denitrification usually does not itself express significant isotopic fractionation (65).Rather, the dominant regional isotopic signal appears to be from compensatory N 2 fixation: Sedimentary denitrification on the shelf tends to stimulate N 2 fixation on a regional basis (57).A reduction in shelf sedimentary denitrification associated with cooling-induced ice growth would tend to reduce regional N 2 fixation, which would decrease its tendency to lower thermocline nitrate δ 15 N in the western North Atlantic.However, for the large sea level drop of ~125 m of the late Pleistocene glacial maxima, foraminifera-bound δ 15 N (FB-δ 15 N) increased by only ~3‰ in the South China Sea (57).Thus, the possible Late Cretaceous sea level decline of 40 m would likely be insufficient to explain the observed 4‰ Campanian-to-Maastrichtian rise in δ 15 N oto .
Given the shortcomings of the regional mechanisms described above, we progress to the interpretation that the Campanianto-Maastrichtian δ 15 N oto increase was due to a baseline δ 15 N increase on the ocean basin and/or global ocean scale.The subsurface nitrate δ 15 N of the whole ocean is sensitive to the proportion of denitrification in the water column versus the sediments (66), with a range of secondary influences (67,68).In this context, the δ 15 N oto increase implies a decrease in sedimentary denitrification and/or an increase in water column denitrification.With the large (~120 m) sea level drop during late Pleistocene glacial maxima, there is no sign that the global mean ocean nitrate δ 15 N rose by anything comparable to the 4‰ increase observed in δ 15 N oto from the Campanian to the Maastrichtian (67).Thus, we tend to discount a global decline in sedimentary denitrification as the cause of the δ 15 N oto increase.
The alternative global ocean explanation for the Campanianto-Maastrichtian δ 15 N oto rise is an increase in water column denitrification, which likely requires expansion of the ocean's suboxic zones.The average oxygen content of ocean water is expected to be lower under warmer climates (69).However, recent model studies suggest that, even as warming causes the global ocean to lose oxygen on average, the thermocline-hosted oxygen-deficient zones (ODZs) can shrink (70,71).The latter change would reduce water column denitrification, lowering nitrate δ 15 N most near the ODZs (today, sited in the eastern tropical Pacific and the Arabian Sea) but also throughout the global pycnocline (68).FB-δ 15 N data from multiple warm periods in the Cenozoic-the Paleogene-Eocene Thermal Maximum, Early Eocene Climate Optimum and Middle Miocene Climate Optimum-are consistent with smaller ODZs under warmer climate.In those warm periods, FB-δ 15 N was substantially lower, with the largest δ 15 N declines occurring in the tropical Pacific, closest to the eastern tropical Pacific ODZs (54,55,72), pointing to ODZ shrinkage (73).While this change would have most strongly reduced nitrate δ 15 N near the shrinking ODZ, it also would have engendered a global ocean nitrate δ 15 N decline because of a decrease in the global ocean's ratio of water column to sedimentary denitrification.Thus, the Campanianto-Maastrichtian δ 15 N oto increase can be explained by the same climate sensitivity of ODZs, with ODZs expanding and water column denitrification accelerating as climate cooled into the Maastrichtian.

Conclusions
From a study of Late Cretaceous deposits along North America's eastern margin, we find that paleoenvironmental signals are preserved in the δ 15 N of fossil fish otoliths as old as ~80 Ma.This opens a new avenue by which to reconstruct paleoenvironmental and paleoecological conditions in Cenozoic and pre-Cenozoic Earth history.For their sustenance, fish ultimately rely on regional phytoplankton production, which records the isotopic composition of fixed N in the environment; thus, fish otoliths provide a potential probe of past changes in the ocean's N cycle.Further, otoliths are diagnostic of specific taxa and can be used to reconstruct the trophic levels of those taxa and potentially the trophic structure of the food web in which they live.In addition, fish otoliths have the potential to fill a gap between fossils from organisms occupying low trophic levels (e.g., planktonic foraminifera and the shells of bivalves and mollusks) and top predators (e.g., shark teeth), all of which can be measured for their fossil-bound N isotopic composition (22,24,26,28,29).Studies of the ocean during the pre-Cenozoic are hampered by the subduction-driven loss of open ocean seafloor over time and the limited evolutionary history of planktonic foraminifera.As fish have a longer evolutionary history and otoliths accumulate in ocean margin sediments, they may prove particularly useful for studies of pre-Cenozoic and even pre-Mesozoic Earth history.
For all three fish taxa analyzed in this study, a δ 15 N oto increase of ~4‰ was observed from the Campanian to the Maastrichtian, a change that we interpret to reflect not trophic level change but rather a baseline δ 15 N increase at the regional and/or global scale.There are several plausible explanations for this baseline δ 15 N change.We favor a change in the nitrate δ 15 N supplied to the North American northeast continental shelf system.This δ 15 N oto change is consistent with the negative correlation between global temperature and ocean oxygen-deficient zone extent that has been reconstructed from foraminifera-bound N isotopes and other proxies from the Cenozoic (54,55,72,73).The observed δ 15 N oto increase may reflect that water column denitrification increased from warmer Campanian to the cooler Maastrichtian.If so, the results indicate that linkages between global climate and ocean oxygen-deficient zone extent were similar between the Mesozoic and Cenozoic Eras, despite the end-Cretaceous mass extinction (74) and changes in ocean basin geometry from the Cretaceous to the Cenozoic (75), suggesting that this biogeochemically and ecologically important sensitivity to global climate is a permanent feature of Earth's ocean.

Materials and Methods
All otoliths were sagittal otoliths, typically the largest of the three paired otoliths in the teleost fishes and the most frequently examined in paleontological and geochemical studies (76).Pollen studies, nannofossils, and foraminiferal biozonation of the Woodbury Formation place it in the early-middle Campanian, with an age of 83.6 to 77.9 Ma (39).The Tar Heel Formation is also dated to early-middle Campanian, based on nannofossils biozones and Sr isotopes (77), and it has similar sedimentologic and stratigraphic features to the Woodbury Formation (42).The Severn Formation represents the Atlantic coastal environment in the Maastrichtian (78).Collected from these formations are otoliths of the three most abundant taxa: E. maastrichtiensis, E. zideki, and Pterothrissus sp.All have been interpreted to inhabit the Cretaceous the upper to middle continental shelf (39,78).We know little about the diet of Eutawichthys spp., as they went extinct at the Cretaceous/Paleogene boundary and do not have typical modern representatives.The closely related modern representative of Pterothrissus sp. is Nemoossis belloci, an endemic species in the eastern Atlantic Ocean (79), is inferred as a nektonic shelf inhabitant that feeds on zooplankton and polychaetes.
Otolith Preservation.The preservation condition of fossil otoliths in our study was evaluated by the presence or absence of salient morphological features.These features are located primarily on the inner face of the sagitta, although some may deal with the entire specimen.Morphological features employed to determine the preservation are listed in SI Appendix, Tables S3 and S4.Specimens with four features or less are designated as poorly preserved, whereas those with six or more are denoted as well preserved.If majorities of the morphological features are present, even if an otolith shows eroded margins and appear to be partially eroded, it could still be deemed as well preserved.Characteristics such as coloration and staining do not affect the evaluated preservation unless they obliterate the morphological features.The morphological features are specific for the otoliths used in this study, namely E. maastrichtiensis and E. zideki.Preservation categories for Pterothrissus sp. were not determined in this study.
Otolith-Bound δ 15 N Nitrogen Isotopic Analysis.All the otoliths were imaged twice under reflective light (Leica KL1500 LCD) with a camera attached to a stereomicroscope (Leica S6D), before and after they underwent an initial cleaning as individual whole otoliths.Each otolith was first cleaned with 10 mL 2% sodium polyphosphate solution under ultrasonication for 10 s.To remove any metal oxide coatings, 10 mL sodium dithionite solution (31 g sodium citrate + 10 g sodium bicarbonate + 25 g sodium hydrosulfite in 500 mL deionized water) was aliquoted into the sample vials, which were placed in an 80 °C water bath for an hour.Finally, to remove external organic contaminants, 10 mL of "bleach" solution (sodium hypochlorite, 10 to 15% available chlorine; Sigma-Aldrich reagent grade) was aliquoted into the sample vials, which were shaken overnight (~15 h).Samples were rinsed with deionized water three times after each cleaning step and were dried at 55 °C.Powdered otolith samples were portioned from the ground otoliths and cleaned again with bleach (or a persulfate reagent in the case of cleaning tests).
The "persulfate-denitrifier" (or "oxidation-denitrifier") method was used for fossil-bound organic matter δ 15 N analysis (19,22,24,30).For each δ 15 N measurement, 2 to 5 mg cleaned otolith powder was weighed into precombusted 4 mL vials.Additionally, 2 to 3 subsamples of in-house coral standard CBS2019 (Diploria labyrinthiformis, 63 to 250 μm grain size) and otolith standard HDS (Melanogrammus aeglefinus, 250 to 425 μm grain size) were analyzed with each sample batch.Each sample or standard was dissolved in 80 μL 4 mol/L HCl to remove the mineral matrix, and 1 mL aliquots of a persulfate-based oxidizing reagent (produced by dissolving 1 g recrystallized potassium persulfate and 2 g sodium hydroxide in 100 mL high-purity deionized water) were then added to each vial to oxidize the organic nitrogen to nitrate (NO 3 − ).Samples were autoclaved under 121 °C for 1 h.After oxidation, all samples were pH-adjusted to 5 to 7, and NO 3 − concentration was measured by conversion to nitric oxide in acidic vanadium solution followed by chemiluminescence detection (80) with a chemiluminescence NO x analyzer (Model 200E, Teledyne Instruments).Samples were then injected into vials containing concentrated culture of a strain of Pseudomonas chlororaphis that lacks nitrous oxide reductase activity to quantitatively convert the NO 3 − to nitrous oxide (N 2 O) (81,82).The δ 15 N of the resulting N 2 O was measured with a custom-built in-line preparation and purification system connected to a gas-source isotope ratio mass spectrometer (GC-IRMS, Thermo Fisher MAT253) (82,83).δ 15 N measurement was corrected for the N blank associated with the persulfate oxidation protocol (SI Appendix).Isotopic ratios of the measured samples are reported relative to the international reference, N 2 in air.
Fossil Otolith Mineralogy Determination.A Fourier transform infrared spectrometer (Vertex 80v, Bruker Corporation) was used to differentiate calcite and aragonite for the fossil otoliths.The IR spectroscopy uses a mercury-cadmiumtelluride detector and measures attenuated total reflection from the sample on a diamond crystal.Powdered sample was placed on the diamond crystal and was compressed to ensure full coverage of the crystal surface with sample materials.Each sample was measured every 2 cm −1 between 600 and 3,998 cm −1 wavenumber and was scanned 500 times to acquire the attenuated reflection.The FTIR spectra were corrected by normalizing the absorbance in reference to the maximum absorbance after subtracting the regression baseline modeled from the 1,800 to 3,998 cm −1 .
Data, Materials, and Software Availability.All study data are included in the article and/or supporting information.
ACKNOWLEDGMENTS.The modern sampling of the US Northeast Continental Shelf was made possible by the NOAA Northeast Fisheries Science Center (NEFSC) Ecosystem Monitoring program, NEFSC Fishery Biology Program, and NEFSC Bottom Trawl Survey.We acknowledge the efforts of NEFSC managers, staff, and shipboard scientists and crew of both programs, with our special appreciation to Sandy Sutherland and Eric Robillard (Fishery Biology Program, Woods Hole, MA) and Jerry Prezioso (NOAA NEFSC, Narragansett, RI).We thank Sheriel Henry, Sam Henry, and Sophia Myers for lab assistance with particulate N and zooplankton.We also thank the Maloof Lab at Princeton University for providing the stereomicroscope for fossil imaging.This work is funded by US NSF grant 10015689 (to D.M.S.), the Scott Fund of the Department of Geosciences, Princeton University, and the Max Planck Society (to G.H.H. and A.M.-G.).J.A.L.-D. was supported by a MarineGEO Postdoctoral Fellowship.We thank Ethan L. Grossman and an anonymous reviewer for their helpful input, which improved this manuscript.

Fig. 1 .
Fig. 1. (A) δ 15 N measurements from the western North Atlantic continental shelf of suspended PN, zooplankton of three size ranges, and butterfish otoliths.(B) Map of the region, with shallow subsurface nitrate δ 15 N as colored circles (from ~150 m depth, or deepest depths along the continental margin).Stars denote the locations of three formations from which the Late Cretaceous otoliths were collected, including, from north to south, the Woodbury Formation (New Jersey, Campanian), Severn Formation (Maryland, Maastrichtian), and Tar Heel Formation (North Carolina, Campanian).The map was generated with ODV (Schlitzer, Reiner, Ocean Data View, odv.awi.de,2023).See SI Appendix for background information on the samples and data in this figure.

Fig. 3 .
Fig. 3. Box plots of otolith-bound δ 15 N and N content measurements for all taxa from Woodbury Formation, Tar Heel Formation and Severn Formation.E. maastrichtiensis results are denoted in yellow squares, E. zideki in red triangles.Filled symbols represent measurements from well-preserved otoliths, open symbols are measurements from poorly preserved otoliths.Green circles are for Pterothrissus sp., the preservation of which were not evaluated.The numbers in parentheses indicate the number of fossil otoliths analyzed.

Fig. 4 .
Fig. 4. Compilation of δ 15 N oto results, plotted with Late Cretaceous sea surface temperature (SST) for the western North Atlantic reconstructed with TEX86 (6).The SST trend is in gray rectangles with dotted-dash line.Colored symbols are average δ 15 N oto of each taxon from each formation (see legend).Vertical error bars are calculated from the SD (1SD) of measured δ 15 N oto .Age constraints (plotted age values and horizontal error bars) of Campanian otoliths are based on estimates from refs.39 and 42.The age of Maastrichtian otoliths uses the age estimates for the entire epoch (sourced from the International Chronostratigraphic Chart).